Alpha-synuclein-containing Lewy bodies are a central pathological feature of Parkinson's disease. Fly, rat and mouse models of alpha-synuclein overexpression have been developed that lead to inclusion formation and toxicity of dopaminergic neural systems. By analogy to studies performed in models of trinucleotide repeat diseases, Bonini and colleagues used the transgenic fly model of alpha-synuclein overproduction and showed dramatic protection against dopaminergic cell death by overexpressing the molecular chaperone, heat shock protein 70 (Hsp70). We now show that Hsp70 and related molecular chaperones stain Lewy bodies in human neuropathological tissue, and overexpression of Hsp70 and cochaperone proteins prevent alpha-synuclein inclusions in an in vitro mammalian cell system. We hypothesize that molecular chaperones play a role in preventing alpha-synuclein misfolding, and perhaps in directing misfolded alpha-synuclein towards degradation. We will study the extent of HSP involvement in Lewy body disease by examining Lewy body tissue using confocal microscopy and the new fluorescent lifetime imaging technique, FLIM, to evaluate the conformation of alpha-synuclein in pathological specimens and relate that to HSP expression. We have found that alpha-synuciein in cells, transgenic mice and human DLB appears in a triton X-100 insoluble fraction; our preliminary data suggest that overexpression of Hsp70 blocks this in cells and transgenic mice. We will extend our in vitro studies of alpha-synuclein aggregation to determine which set of HSP cochaperones are most effective at preventing alpha-synuciein inclusions and toxicity, and whether down-regulation of HSPs leads to increased inclusions and toxicity. Finally, we will use transgenic animals and new gene transfer techniques to determine the effects of overexpression of HSP on alpha-synuclein inclusions and dopaminergic terminal toxicity observed in a transgenic mouse overexpressing alpha-synuclein, Line D. Taken together we propose an integrated set of aims to study the role of HSPs in alpha-synuclein aggregation and toxicity.These projects also integrate tightly with the aims described in the Lindquist, Graybiel and Standaert projects.